Introduction and characterization of a polymerase-dead point mutation into the POLK gene in vertebrates

Takenaka, Katsuya; Miki, Yoshio

doi:10.1016/j.febslet.2008.12.057

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Volume 583, Issue 4 , Pages 661-664, 18 February 2009

Introduction and characterization of a polymerase-dead point mutation into the POLK gene in vertebrates

Edited by Varda Rotter

Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo, 113-8510 Tokyo, Japan

Received 2 October 2008; received in revised form 21 December 2008; accepted 24 December 2008. published online 21 January 2009.

Abstract

The chicken DT40 cell line is widely used for gene knock-outs. We attempted to introduce a polymerase-dead point mutation into Polκ, a polymerase for translesion DNA synthesis, taking advantage of the highly efficient targeted integration in DT40 cells. The resulting cells (REV3^−/−POLK^/^pol-dead) proliferated with the same kinetics as the parental REV3^−/− cells. Though the mock-treated REV3^−/−POLK^/^mock cells showed the same sensitivity as the parental REV3^−/− cells to methyl methanesulfonate, the REV3^−/−POLK^/^pol-dead cells demonstrated the same sensitivity as the REV3^−/−POLK^/− double knock-out cells. This implies that the presence of the polymerase-dead Polκ does not interfere with other polymerases repairing monoalkylation damage.